1. Field of the Invention
The present invention relates to a specific nucleic sequence of Campylobacter coli, as well as to applications of this sequence as specific nucleotide probe for the detection of sequences of Campylobacter coli or of fragments of this sequence as nucleotide primers for the amplification of DNA or RNA of Campylobacter coli in a biological specimen.
2. Description of the Background Art
Campylobacter infections are very common all over the world, affecting man as well as wild or domestic animals.
Although discovered at the start of the twentieth century, the bacteria now called Campylobacter were unrecognized for a long time, because their characteristics rendered their identification and their culture difficult. First isolated in sheep and cattle and called Vibrio fetus and then, later, Campylobacter fetus, it was not until the start of 1946 that the first cases of human campylobacterioses were described, but it was not until the start of 1972, when selective media for Campylobacter had begun to be perfected, that the importance of Campylobacter infections had been able to be proved and recognized.
Since the naming of the species type Campylobacter fetus, about a dozen other species and subspecies have been discovered, the exact number varying according to the authors and the taxonomic methods, which often propose novel classification criteria. Among these species, the most frequently encountered in human and/or animal pathology are Campylobacter jejuni, Campylobacter coli and Campylobacter fetus.
At present, Campylobacter jejuni and Campylobacter coli are frequently responsible for infectious diarrhoea in man.
The "national surveillance system of Campylobacter infections", set up in France in 1986, publishes each year an assessment making evident the principal epidemiological and clinical data of the cases reported.
In the human species, the major symptom of intestinal infection with C. jejuni or with C. coli is diarrhoea which, in the most serious cases, can involve serious losses of water, which can be particularly dangerous for children and infants, who are very sensitive to dehydration. However, Campylobacter enteritis is often without complications and diarrhoeas can even cease spontaneously at the end of one week. However, the coprocultures can remain positive up to several weeks or even months, and, in 5 to 10% of the cases, relapses can occur. Treatment and rigorous follow-up are then essential, especially for subjects who are immunodepressed or have serious disorders (AIDS, cirrhosis of the liver, cancer, leukaemia, etc.), in which Campylobacter can behave like opportunistic bacteria.
Other consequences of C. jejune or C. coli infections have also been described, although rarer or more exceptional: mesenteric adenitis, cholecystitis, urinary infections, meningites, septicaemias, erythema nodosum or Guillain-Barre syndrome, etc.
In animals, the Campylobacter usually live in commensals in the alimentary canal of numerous species: cattle, sheep, pigs, poultry, wild birds, dogs and cats. These animals, whether ill or healthy carriers, form a large reservoir of germs, and thus an important risk of contamination. In the case of obvious infections, in cattle and sheep, C. jejuni is known, since the first description in 1931, as being the cause of "bovine dysentery", which can have the result, apart from the effect on the cattle, of transmission to man through the dissemination of germs in the environment of the animals (ground, water). Even for the asymptomatic animals, "healthy carriers", transmission to man can take place: either by direct contact with these animals or their excrement, or by consumption of contaminated food or water (meats contaminated during their preparation and badly cooked, non-pasteurized milk, polluted water, etc.).
From a prevention viewpoint, it is important then, both in man and in animals, to be able to identify the pathogen C. jejuni or C. coli as soon as possible, in order to prevent, by means of adequate measures, any contamination. This is particularly the case in the farm-produce industry, where conditions of sterility must be respected. It is equally important in human pathology, to carry out a proper follow-up of the disorders treated following a Campylobacter infection, in order to avoid any new relapse.
Finally, in the case of notified infections, it is very important to identify properly the germ responsible, and to do this rapidly after the triggering of the disorder, in order to be able to administer a proper and efficacious treatment which will prevent the progress of the infection, or even the growth of epidemics. However, the identification of the Campylobacter and the determination of the incriminated species is not easy. In fact, their isolation requires special media and their conventional detection does not take place at present until after an enrichment by culturing for at least 48 hours. This is very long when a rapid diagnosis is necessary. On the other hand, given that the microbiological diagnosis takes place at present by bacteriological and/or biochemical techniques which exploit phenotype differences existing between the different species, errors of diagnosis can occur, especially when mutants appear for a given characteristic. In the distinct case of C. jejuni and C. coli, the unique criterion of differentiation is hydrolysis of hippurate (C. jejuni can hydrolyse it although C. coli cannot), and it sometimes happens that this distinction cannot be made because there are hippurate-negative C. jejuni strains (Hebert et al., J. Clin. Microbiol., 1984, 20, 138-104, Totten et al., J. Clin. Microbiol., 1987, 25, 1747-1752).
Approaches using molecular hybridization to identify the strains belonging to the genus Campylobacter have been proposed. However, these methods do not allow identification until after culture; they are not sufficiently sensitive to detect this bacterium in the biological samples. Thus, methods of identification and of classification of Campylobacter have been proposed using either radioactive probes (Ng et al., Mol. Cell. Probes, 1987, 1, 233-243), or non-radioactive probes (Chevrier et al., J. Clin. Microbiol., 1989, 27, 321-326), but these methods use total genomic probes and necessitate enrichment by culture of the pathogen to be detected, because the threshold of detection is sufficiently high, approximately 10.sup.5 bacteria (Chevrier et al., above).
Research on specific nucleic probes of C. jejuni, with an aim of diagnosis of species, has been performed by Picken et al., (Mol. Cell. Probes, 1987, 1, 245-259), Korolik et al., (J. Gen. Microbiol., 1988, 134, 521-529) and Zhou and Wang (Zbl. Bakt., 1989, 272, 186-190), but there remain problems of specificity and the sequences of these potential probes have not been determined.
Recently the inventors, the authors of the present invention, have discovered a specific DNA sequence of the species C. jejuni (FR-2 701 028). This sequence has been used to perfect a PCR test capable of detecting and identifying this species in a biological specimen (Stonnet and Guesdon, FEMS Immunol. Med. Microbiol., 1993, 7, 337-344). Taking account of the high specificity of the sequence, this PCR test does not allow the species C. coli, which is just as important clinically as C. jejuni to be detected. Recently, a specific method of identification and of discrimination among the thermophilic Campylobacter, including C. coli, has been described by Eyers et al., (J. Clin. Microbiol., 1993, 31, 3340-3343). However, the specific primers of species have been chosen in hypervariable regions of the genes coding for 23S ribosomal RNA, which does not exclude the possibility of obtaining "false-negative" results when the DNA of a strain to be tested possesses variations just at the sequence of the primers.